SUMMARY/ABSTRACT The development of drugs for Neglected Tropical Diseases (NTDs) suffers from a critical gap in the discovery process, wherein drugs screened in vitro rarely progress to the preclinical phase. While academic research laboratories often discover new compounds effective in vitro, development organizations are reluctant to perform the pre-clinical research until lead compounds show proven activity in appropriate animal models. We propose to fill this gap for three NTDs: cutaneous leishmaniasis (CL), visceral leishmaniasis (VL), and Chagas disease by contributing new, high quality chemical matter to the candidate-seeking pipeline. This will be accomplished by producing compounds that are effective in the in vivo models of the diseases and that meet target-product profiles as described by the Drugs for Neglected Diseases initiative (DNDi). We have produced hundreds of compounds that have been tested against T. cruzi (n=312) and Leishmania major (n=420), and have identified 83 anti-chagasic compounds (EC50 ? 5 M), 127 L. major inhibitors (EC50 ? 5 M), and 53 inhibitors with potent activity against both pathogens. The key limitations of these compounds are in their metabolic and physical properties: limited solubility, high clearance, and high plasma protein binding. We also have in vitro data against only one species of Leishmania (L. major, which causes CL). To address these limitations we propose in the R21 phase of this project to prioritize the compounds and to identify key weaknesses that can be addressed by systematic medicinal chemistry in the R33 phase. The prioritization will require completion of in vitro L. donovani assays (of relevance to VL), and prioritization of compounds for mouse pharmacokinetic (PK) experiments. Once compounds with acceptable potency and PK properties are identified from the existing set of analogs, prioritized compounds will be advanced to assessment in mouse infection models of all three diseases. Those compounds that show efficacy in animal models of infection will be profiled in toxicity assays (human kinase, GPCR, ion channel activity, genotoxicity), completing a data package that will drive the later medicinal chemistry optimization program. In order to transition to the R33 phase, we will have minimally identified at least two compounds that meet the defined lead criteria for at least one pathogen assessed, with key issues identified for optimization in the R33 phase of the project. This phase of the project will be focused on lead-to-preclinical candidate medicinal chemistry optimization in order to achieve the targeted properties for further advancement. In order to accomplish these goals, we will employ a unique combination of academic, government, and industry collaborators. By the end of the R33 phase, we will deliver compounds that effect parasitological cure in animal models of Chagas disease, VL, and/or CL using the dosing paradigm and endpoints required by DNDi. With NIAID Therapeutic Development Services, we will round out the necessary data package for a candidate compound and one backup compound in order to either make a go/no-go decision for continued advancement towards clinical trials.